Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils — neutrophil extracellular traps (NETs), in particular — in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1–KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1–deficient mice by infusion of WT neutrophils, while an anti–PSGL-1 monoclonal antibody inhibited APS IgG–mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.
Jason S. Knight, He Meng, Patrick Coit, Srilakshmi Yalavarthi, Gautam Sule, Alex A. Gandhi, Robert C. Grenn, Levi F. Mazza, Ramadan A. Ali, Paul Renauer, Jonathan D. Wren, Paula L. Bockenstedt, Hui Wang, Daniel T. Eitzman, Amr H. Sawalha
The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12–deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.
Priscilla W. Lee, Alan J. Smith, Yuhong Yang, Amanda J. Selhorst, Yue Liu, Michael K. Racke, Amy E. Lovett-Racke
Recent data indicate that there are different subpopulations of Th17 cells that can express a regulatory as opposed to an inflammatory gene signature. The transmembrane glycoprotein PDPN is critical in the development of multiple organs including the lymphatic system and has been described on T cells in mouse models of autoimmune Th17 inflammation. Here, we demonstrate that unlike in mice, PDPN+ T cells induced under classic Th17-polarizing conditions express transcription factors associated with Th17 cells but do not produce IL-17. Moreover, these cells express a transcriptional profile enriched for immunosuppressive and regulatory pathways and express a distinct cytokine profile compared with potentially pathogenic PDPN– Th17 cells. Ligation of PDPN by its ligand CLEC-2 ameliorates the Th17 inflammatory response. IL-17 secretion is restored with shRNA gene silencing of PDPN. Furthermore, PDPN expression is reduced via an Sgk1-mediated pathway under proinflammatory, high sodium chloride conditions. Finally, CD3+PDPN+ T cells are devoid of IL-17 in skin biopsies from patients with candidiasis, a prototypical Th17-driven skin disease. Thus, our data support the hypothesis that PDPN may serve as a marker of a nonpathogenic Th17 cell subset and may also functionally regulate pathogenic Th17 inflammation.
Alyssa N. Nylander, Gerald D. Ponath, Pierre-Paul Axisa, Mayyan Mubarak, Mary Tomayko, Vijay K. Kuchroo, David Pitt, David A. Hafler
The role of B cells and posttranslational modifications in pathogenesis of organ-specific immune diseases is increasingly envisioned but remains poorly understood, particularly in human disorders. In celiac disease, transglutaminase 2–modified (TG2-modified; deamidated) gluten peptides drive disease-specific T cell and B cell responses, and antibodies to deamidated gluten peptides are excellent diagnostic markers. Here, we substantiate by high-throughput sequencing of IGHV genes that antibodies to a disease-specific, deamidated, and immunodominant B cell epitope of gluten (PLQPEQPFP) have biased and stereotyped usage of IGHV3-23 and IGHV3-15 gene segments with modest somatic mutations. X-ray crystal structures of 2 prototype IGHV3-15/IGKV4-1 and IGHV3-23/IGLV4-69 antibodies reveal peptide interaction mainly via germline-encoded residues. In-depth mutational analysis showed restricted selection and substitution patterns at positions involved in antigen binding. While the IGHV3-15/IGKV4-1 antibody interacts with Glu5 and Gln6, the IGHV3-23/IGLV4-69 antibody interacts with Gln3, Pro4, Pro7, and Phe8 — residues involved in substrate recognition by TG2. Hence, both antibodies, despite different interaction with the epitope, recognize signatures of TG2 processing that facilitates B cell presentation of deamidated gluten peptides to T cells, thereby providing a molecular framework for the generation of these clinically important antibodies. The study provides essential insight into the pathogenic mechanism of celiac disease.
Omri Snir, Xi Chen, Moriah Gidoni, M. Fleur du Pré, Yuguang Zhao, Øyvind Steinsbø, Knut E.A. Lundin, Gur Yaari, Ludvig M. Sollid
Myasthenia gravis (MG) is a B cell–mediated autoimmune disorder of neuromuscular transmission. Pathogenic autoantibodies to muscle-specific tyrosine kinase (MuSK) can be found in patients with MG who do not have detectable antibodies to the acetylcholine receptor (AChR). MuSK MG includes immunological and clinical features that are generally distinct from AChR MG, particularly regarding responsiveness to therapy. B cell depletion has been shown to affect a decline in serum autoantibodies and to induce sustained clinical improvement in the majority of MuSK MG patients. However, the duration of this benefit may be limited, as we observed disease relapse in MuSK MG patients who had achieved rituximab-induced remission. We investigated the mechanisms of such relapses by exploring autoantibody production in the reemerging B cell compartment. Autoantibody-expressing CD27+ B cells were observed within the reconstituted repertoire during relapse but not during remission or in controls. Using two complementary approaches, which included production of 108 unique human monoclonal recombinant immunoglobulins, we demonstrated that antibody-secreting CD27hiCD38hi B cells (plasmablasts) contribute to the production of MuSK autoantibodies during relapse. The autoantibodies displayed hallmarks of antigen-driven affinity maturation. These collective findings introduce potential mechanisms for understanding both MuSK autoantibody production and disease relapse following B cell depletion.
Panos Stathopoulos, Aditya Kumar, Richard J. Nowak, Kevin C. O’Connor
Human endogenous retroviruses (HERVs), remnants of ancestral viral genomic insertions, are known to represent 8% of the human genome and are associated with several pathologies. In particular, the envelope protein of HERV-W family (HERV-W-Env) has been involved in multiple sclerosis pathogenesis. Investigations to detect HERV-W-Env in a few other autoimmune diseases were negative, except in type-1 diabetes (T1D). In patients suffering from T1D, HERV-W-Env protein was detected in 70% of sera, and its corresponding RNA was detected in 57% of peripheral blood mononuclear cells. While studies on human Langerhans islets evidenced the inhibition of insulin secretion by HERV-W-Env, this endogenous protein was found to be expressed by acinar cells in 75% of human T1D pancreata. An extensive immunohistological analysis further revealed a significant correlation between HERV-W-Env expression and macrophage infiltrates in the exocrine part of human pancreata. Such findings were corroborated by in vivo studies on transgenic mice expressing HERV-W-env gene, which displayed hyperglycemia and decreased levels of insulin, along with immune cell infiltrates in their pancreas. Altogether, these results strongly suggest an involvement of HERV-W-Env in T1D pathogenesis. They also provide potentially novel therapeutic perspectives, since unveiling a pathogenic target in T1D.
Sandrine Levet, Julie Medina, Julie Joanou, Amandine Demolder, Nelly Queruel, Kevin Réant, Matthieu Normand, Marine Seffals, Julie Dimier, Raphaële Germi, Thomas Piofczyk, Jacques Portoukalian, Jean-Louis Touraine, Hervé Perron
The pathogenesis of primary Sjogren’s syndrome (SS), an autoimmune disease that targets the mucosa of exocrine tissues, is poorly understood. Although several mouse models have been developed that display features of SS, most of these are within the larger context of a lupus-like presentation. Immunity-related GTPase family M protein 1 (Irgm1) is an interferon-inducible cytoplasmic GTPase that is reported to regulate autophagy and mitochondrial homeostasis. Here, we report that naive Irgm1–/– mice display lymphocytic infiltration of multiple mucosal tissues including the lung in a manner reminiscent of SS, together with IgA class–predominant autoantibodies including anti-Ro and anti-La. This phenotype persists in the germ-free state, but is abolished by deletion of Irgm3. Irgm1–/– mice have increased local production in the lung of TECP15-idiotype IgA, a natural antibody with dual reactivity against host and pneumococcal phosphorylcholine. Associated with this, Irgm1–/– mice display enhanced opsonization and clearance of Streptococcus pneumoniae from the lung and increased survival from pneumococcal pneumonia. Taken together, our results identify Irgm1 as a master regulator of mucosal immunity that dually modulates evolutionarily conserved self- and other-directed immune responses at the interface of host with environment.
Kathleen M. Azzam, Jennifer H. Madenspacher, Derek W. Cain, Lihua Lai, Kymberly M. Gowdy, Prashant Rai, Kyathanahalli Janardhan, Natasha Clayton, Willie Cunningham, Heather Jensen, Preeyam S. Patel, John F. Kearney, Gregory A. Taylor, Michael B. Fessler
The maintenance of peripheral naive T lymphocytes in humans is dependent on their homeostatic division, not continuing emigration from the thymus, which undergoes involution with age. However, postthymic maintenance of naive T cells is still poorly understood. Previously we reported that recent thymic emigrants (RTEs) are contained in CD31+CD25− naive T cells as defined by their levels of signal joint T cell receptor rearrangement excision circles (sjTRECs). Here, by differential gene expression analysis followed by protein expression and functional studies, we define that the naive T cells having divided the least since thymic emigration express complement receptors (CR1 and CR2) known to bind complement C3b- and C3d-decorated microbial products and, following activation, produce IL-8 (CXCL8), a major chemoattractant for neutrophils in bacterial defense. We also observed an IL-8–producing memory T cell subpopulation coexpressing CR1 and CR2 and with a gene expression signature resembling that of RTEs. The functions of CR1 and CR2 on T cells remain to be determined, but we note that CR2 is the receptor for Epstein-Barr virus, which is a cause of T cell lymphomas and a candidate environmental factor in autoimmune disease.
Marcin L. Pekalski, Arcadio Rubio García, Ricardo C. Ferreira, Daniel B. Rainbow, Deborah J. Smyth, Meghavi Mashar, Jane Brady, Natalia Savinykh, Xaquin Castro Dopico, Sumiyya Mahmood, Simon Duley, Helen E. Stevens, Neil M. Walker, Antony J. Cutler, Frank Waldron-Lynch, David B. Dunger, Claire Shannon-Lowe, Alasdair J. Coles, Joanne L. Jones, Chris Wallace, John A. Todd, Linda S. Wicker
The chemokine receptor CCR6 marks subsets of T cells and innate lymphoid cells that produce IL-17 and IL-22, and as such may play a role in the recruitment of these cells to certain inflammatory sites. However, the precise role of CCR6 has been controversial, in part because no effective monoclonal antibody (mAb) inhibitors against this receptor exist for use in mouse models of inflammation. We circumvented this problem using transgenic mice expressing human CCR6 (hCCR6) under control of its native promoter (hCCR6-Tg/mCCR6–/–). We also developed a fully humanized mAb against hCCR6 with antagonistic activity. The expression pattern of hCCR6 in hCCR6-Tg/mCCR6–/– mice was consistent with the pattern observed in humans. In mouse models of experimental autoimmune encephalomyelitis (EAE) and psoriasis, treatment with anti-hCCR6 mAb was remarkably effective in both preventive and therapeutic regimens. For instance, in the imiquimod model of psoriasis, anti-CCR6 completely abolished all signs of inflammation. Moreover, anti-hCCR6 attenuated clinical symptoms of myelin oligodendrocyte glycoprotein–induced (MOG-induced) EAE and reduced infiltration of inflammatory cells in the central nervous system. CCR6 plays a critical role in Th17 type inflammatory reactions, and CCR6 inhibition may offer an alternative approach for the treatment of these lesions.
Remy Robert, Caroline Ang, Guizhi Sun, Laurent Juglair, Ee X. Lim, Linda J. Mason, Natalie L. Payne, Claude C.A. Bernard, Charles R. Mackay
GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord–infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1P1, and LPHN2 as examples, we show how this information can be used to develop new strategies for the functional modulation of Th17 cells and activated endothelial cells. Taken together, single-cell GPCR expression analysis identifies functionally relevant subpopulations with specific GPCR repertoires and provides a basis for the development of new therapeutic strategies in immune disorders.
Denise Tischner, Myriam Grimm, Harmandeep Kaur, Daniel Staudenraus, Jorge Carvalho, Mario Looso, Stefan Günther, Florian Wanke, Sonja Moos, Nelly Siller, Johanna Breuer, Nicholas Schwab, Frauke Zipp, Ari Waisman, Florian C. Kurschus, Stefan Offermanns, Nina Wettschureck
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